Our recent work has focused on two main aims. First, we have developed methodology to better address and understand neural crest development, including single cell level analysis. Our recently published work presents in vitro culture conditions that are required to maintain trunk neural crest stem cells as self-renewing crestospheres without spontaneous differentiation. Such conditions are a useful tool study mechanisms of trunk neural crest stem cell properties both in terms of transcriptional profiling and functional assays to address stem cell properties such as ability to self-renew or differentiate. These conditions are modified from our previous work, which defined the optimal conditions for cranial crestospheres and highlight the differences between neural crest cells derived from different axial levels of the developing chick embryo. The trunk level is relevant in understanding formation of certain neurocristopathies such as the pediatric cancer neuroblastoma. We have also focused on further development of methodology to enable analysis and subgrouping of cells in intact tissues based on their volumetric differences and morphology. This computational method aims to provide a useful tool for phenotype analysis for broad usage within life-sciences, to complement information acquired from imaging, which currently predominantly is based on analysis of fluorescent biomarkers. Second, we have focused on studies that aim to understand how the neural crest stem cell niche is formed, both by using chicken embryos and human embryonic stem cell derived neural crest cells as our model system. Our ongoing studies include stage by stage single cell level analysis by using various approaches ranging from multiplex in situ hybridization to advanced protein biochemistry and functional knockdown experiments and phenotype analysis.